Historically, protein purification schemes have been predicated on differences in the molecular properties of size, charge and solubility between the protein to be purified and undesired protein contaminants. Protocols based on these parameters include size exclusion chromatography, ion exchange chromatography, differential precipitation and the like.
Size exclusion chromatography, otherwise known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration is a function of the hydrodynamic volume of the particles. Accordingly, under ideal conditions the larger molecules are excluded from the interior of the particles while the smaller molecules are accessible to this volume and the order of elution can be predicted by the size of the protein because a linear relationship exists between elution volume and the log of the molecular weight.
Chromatographic supports based on cross-linked dextrans e.g. SEPHADEX®, spherical agarose beads e.g. SEPHAROSE® (both commercially available from Pharmacia AB. Uppsala, Sweden), based on cross-linked polyacrylamides e.g. BIO-GEL ® (commercially available from BioRad Laboratories, Richmond, Calif.) or based on ethylene glycol-methacrylate copolymer e.g. TOYOPEARL HW65 (commercially available from Toso Haas Co., Tokyo, Japan) are useful in forming the various chromatographic columns for size exclusion, or HIC chromatography in the practice of certain aspects of this invention.
Precipitation methods are predicated on the fact that in crude mixtures of proteins the solubilities of individual proteins are likely to vary widely. Although the solubility of a protein in an aqueous medium depends on a variety of factors, for purposes of this discussion it can be said generally that a protein will be soluble if its interaction with the solvent is stronger than its interaction with protein molecules of the same or similar kind. Without wishing to be bound by any particular mechanistic theory describing precipitation phenomena, it is nonetheless believed that interaction between a protein and water molecules can occur by hydrogen bonding with several types of uncharged groups and/or electrostatically, as dipoles, with charged groups and that precipitants such as salts of monovalent cations (e.g. ammonium sulfate) compete with proteins for water molecules. Thus at high salt concentrations, the proteins become “dehydrated” reducing their interaction with the aqueous environment and increasing the aggregation with like or similar proteins, resulting in precipitation from the medium.
Ion exchange chromatography involves the interaction of charged functional groups in the sample with ionic functional groups of opposite charge on an adsorbent surface. Two general types of interaction are known. Anionic exchange chromatography is mediated by negatively charged amino acid side chains (e.g., aspartic acid and glutamic acid) interacting with positively charged surfaces and cationic exchange chromatography is mediated by positively charged amino acid residues (e.g., lysine and arginine) interacting with negatively charged surfaces.
More recently affinity chromatography and hydrophobic interaction chromatography techniques have been developed to supplement the more traditional size exclusion and ion exchange-chromatographic protocols. Affinity chromatography relies on the specific interaction of the protein with an immobilized ligand. The ligand can be specific for the particular protein of interest in which case the ligand is a substrate, substrate analog, inhibitor, receptor or antibody. Alternatively, the ligand may be able to react with a number of related proteins. Such group specific ligands as adenosine monophosphate, adenosine diphosphate, nicotine adenine dinucleotide or certain dyes may be employed to recover a particular class of proteins.
With respect to the purification of antibody molecules, both specific and generalized affinity techniques are applicable. The most specific choice of ligand for the affinity purification of an antibody is the antigen (or an epitope thereof) to which desired antibody reacts. Many of the well-known immunosorbent assays such as the enzyme-linked immunosorbent assays (ELISA) are predicated on such specific antigen/antibody affinity interactions.
However, generalized affinity techniques are also useful. For example, Staphylococcal Protein A is known to bind certain antibodies of the IgG class (See: Ey, P. L. et al. Immunochemistry 15:429-36 (1978)). Alternatively, antisera raised in heterologous species (e.g. rabbit anti-mouse antisera) can be used to separate general groups of antibodies. (See, Current Protocols in Molecular Biology Supra, Chap 11.)
Hydrophobic interaction chromatography was first developed following the observation that proteins could be retained on affinity gels which comprised hydrocarbon spacer arms but lacked the affinity ligand. Although in this field the term hydrophobic chromatography is sometimes used, the term hydrophobic interaction chromatography (HIC) is preferred because it is the interaction between the solute and the gel that is hydrophobic not the chromatographic procedure. Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures. Elution from HIC supports can be effected by alterations in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene or propylene glycol. A description of the general principles of hydrophobic interaction chromatography can be found in U.S. Pat. No. 3,917,527 and in U.S. Pat. No. 4,000,098. The application of HIC to the purification of specific proteins is exemplified by reference to the following disclosures: human growth hormone (U.S. Pat. No. 4,332,717), toxin conjugates (U.S. Pat. No. 4,771,128), antihemolytic factor (U.S. Pat. No. 4,743,680), tumor necrosis factor (U.S. Pat. No. 4,894,439), interleukin-2(U.S. Pat. No. 4,908,434), human lymphotoxin (U.S. Pat. No. 4,920,196) and lysozyme species (Fausnaugh, J. L. and F. E. Regnier, J. Chromatog. 359:131-146 (1986)) and soluble complement receptors (U.S. Pat. No. 5,252,216). HIC in the context of high performance liquid chromatography (HPLC) has been used to separate antibody fragmens (e.g., F(ab′)2) from intact antibody molecules in a single step protocol. (Morimoto, K. et al., J. Biochem. Biophys. Meth. 24:107-117 (1992)).
In addition to affinity and HIC techniques, one or more of the traditional protein purification schemes have been applied to antibody purification. For example, Hakalahti, L. et al., (J. Immunol. Meth. 117:131-136 (1989)) disclose a protocol employing two successive ion exchange chromatographic steps or one employing a single ion exchange step followed by a HIC step. Danielsson A. et al. (J. Immunol. Methods 115:79-88 (1988)) compare single step protocols based on anion exchange, cation exchange, chromatofocusing and HIC respectively.
Although Protein A affinity column chromatography is widely used, it is also appreciated that elution of antibody from such columns can result in leaching of residual Protein A from the support. Size exclusion HPLC (Das et al., Analytical Biochem, 145:27-36 (1985)) and anion exchange chromatography (EPO345549, published Dec. 13, 1989) have been suggested as means for dealing with this problem.
It has now been surprisingly discovered that HIC can be usefully employed to remove contaminating Protein A from IgG mixtures eluted from Protein A chromatographic support.
This invention relates to the application of HIC to the separation of monomeric IgG from mixtures containing same and to the integration of HIC into a protocol combining Protein A and ion exchange chromatography for the purification of immunoglobulin G molecules.